Graft-versus-host disease (GvHD) is a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation. However, the pathophysiology of GvHD remains poorly understood. In this study, we analyzed the induction of Th17 cells by monocytes of patients with GvHD in vitro, demonstrating that monocytes isolated from patients with acute skin and intestinal GvHD stage I–IV and chronic GvHD induce significantly increased levels of Th17 cells compared with patients without GvHD. S100 proteins are known to act as innate amplifier of inflammation. We therefore investigated the presence of S100 proteins in the stool, serum, and bowel tissue of patients with GvHD and the influence of S100 proteins on the induction of Th17 cells. Elevated levels of S100 proteins could be detected in patients with acute GvHD, demonstrating the release of these phagocyte-specific proteins during GvHD. Furthermore, stimulation of monocytes with S100 proteins was found to promote Th17 development, emphasizing the role of S100 proteins in Th17-triggered inflammation. Altogether, our results indicate that induction of Th17 cells by activated monocytes and the stimulatory effects of proinflammatory S100 proteins might play a relevant role in the pathogenesis of acute GvHD. Regarding our data, S100 proteins might be novel markers for the diagnosis and follow-up of GvHD.

Combinatory strategies are becoming of increasing interest in cancer immunotherapy. Costimulation by individual members of the immunoglobulin-like (Ig)- and TNF superfamily have already shown promising antitumor potential, thus prompting the exploration of their synergistic abilities in combinatorial approaches. Here, we pursued a targeted strategy with antibody-fusion proteins composed of a tumor-directed antibody and the extracellular domain of the costimulatory ligand B7.1, 4-1BBL, OX40L, GITRL or LIGHT, respectively. Costimulatory activity was assessed in an experimental setting where initial T cell activation was induced by a bispecific antibody (tumor-related antigen × CD3). Advantage of combined targeted costimulation was shown for either B7.1 or 4-1BBL with OX40L, GITRL, LIGHT and 4-1BBL in terms of T cell proliferation and IFN-γ release. Since encouraging results were obtained by the combination of B7.1 and 4-1BBL, we adapted the model system for a time-shift setting. Here, enhanced proliferation and granzyme B expression as well as reduced PD-1 expression on the T cell population demonstrated the benefit of costimulation-assisted restimulation. Finally, the antitumor potential of this combinatorial setting was confirmed in vivo in a lung metastasis mouse model. Thus, combinatorial approaches with costimulatory antibody-ligand fusion proteins seem a promising strategy to be further investigated for cancer immunotherapy.

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Graft-versus-host disease (GvHD) is a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). The pathophysiology of GvHD is complex and remains poorly understood. However, it has been demonstrated that Th17 cells play an important role in the development and progression of acute and chronic GvHD. Furthermore, monocytes have an essential function in the induction of Th17 cells. It is known that the distribution of the three monocyte subtypes (classical (CD14++ CD16-), non-classical (CD14+ CD16++) and intermediate (CD14++ CD16+) monocytes) changes significantly in the peripheral blood of patients during GvHD: the level of classical monocytes is decreased, whereas the percentages of intermediate and non-classical monocytes are elevated. Additionally, there is first evidence that intermediate monocytes are expanded during rheumatoid arthritis and promote the expansion of Th17 cells. Recently it could be demonstrated that pro-inflammatory Th17 cells are restricted to a novel subset of CCR6+ CXCR3hi CCR4lo CCR10- CD161+ cells that stably express P-glycoprotein/multi-drug resistance protein type 1 (MDR1) - the so-called Th17.1 cells. These MDR1+ Th17.1 cells are enriched and activated in the gut of patients with Crohn's disease and are resistant to glucocorticoid-mediated T cell suppression. Thus, this subtype of Th17 cells might play an important role in steroid-resistant inflammatory diseases as well as steroid-refractory GvHD. Our previously performed study could demonstrate that monocytes isolated from patients with acute or chronic GvHD grade I-IV (n=13) induced significant higher percentages of IL-17-producing Th17 cells compared to monocytes from healthy donors (n=32) or patients without GvHD after HCT (n=21) (p<0.001). To further investigate the role of monocytes in the pathogenesis of GvHD the influence of classical, non-classical and intermediate monocytes on the induction of pro-inflammatory MDR1+ CCR6+ CXCR3hi CCR4lo CCR10- CD161+ Th17.1 cells was investigated in the present study. Therefore, these monocyte subtypes were isolated from peripheral blood mononuclear cells (PBMCs) by magnetic cell isolation technology and were co-cultured with isolated CD4+ T cells. The obtained results could show that intermediate and non-classical monocytes induced higher percentages of pro-inflammatory MDR1+ CCR6+ CXCR3hi CCR4lo CCR10- CD161+ Th17.1 cells compared to classical monocytes or whole monocyte population. Additionally, the influence of glucocorticoids used for the treatment of patients with GvHD and the Hsp90 inhibitor 17-Dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) was examined on the development of MDR1 expressing Th17.1 cells in vitro. Therefore, monocytes were co-cultured with CD4+ T cells in the presence and absence of prednisolone, dexamethasone and 17-DMAG. First data demonstrated that treatment of these co-cultures with glucocorticoids resulted in elevated levels of induced pro-inflammatory MDR1+ Th17.1 cells. In contrast, addition of the Hsp90 inhibitor 17-DMAG to the co-cultures led to decreased levels of these pro-inflammatory Th17 cells compared to the untreated control. In conclusion, our findings suggest that intermediate and non-classical monocytes trigger the induction of pro-inflammatory MDR1+ CCR6+ CXCR3hi CCR4lo CCR10- CD161+ Th17.1 cells and might therefore play a major role in the pathogenesis of GvHD. While glucocorticoids seem to promote the development of MDR1 expressing Th17.1 cells, the Hsp90 inhibitor 17-DMAG acts anti-inflammatory leading to decreased levels of induced pro-inflammatory Th17 cells. Thus, the chaperone Hsp90 could be an important regulator in monocyte-induced development of pro-inflammatory MDR1+ Th17.1 cells and might therefore be a therapeutic target for GvHD. Disclosures No relevant conflicts of interest to declare.